How oxygen-limitation shapes the efficiency of key promoters for recombinant protein production in Komagataella phaffii

Komagataella phaffii (formerly Pichia pastoris ) is a well-established host for recombinant protein production (RPP), valued for its robustness and scalability in industrial bioprocesses. Improvements in bioprocess efficiency do not depend solely on strain engineering, such as promoter selection and/or engineering, but rather on the appropriate combination of promoter choice and process design strategy. Among critical factors, oxygen-limiting conditions are common in large-scale fermentations, making it particularly relevant to understand how promoters respond to this type of physiological stress. Previous studies have shown that the P GAP promoter can experience a significant increase in titers and productivity under hypoxia; therefore, in this work, we aimed to evaluate whether this effect extends to other widely used promoters from the K. phaffii expression toolbox. In this study, the effect of hypoxic conditions on the production of Candida antarctica lipase B (CalB) as a model protein was systematically evaluated under the regulation of six different methanol-free promoters ( P DF , P UPP , P PGK1 , P DC , P DH , P GAP ). CalB was selected given its significant interest as an industrial enzyme, not only for hydrolysis reactions but also for synthesis applications. Additionally, a recombinant strain expressing CalB under the classical methanol-inducible promoter P AOX1 was used as a benchmark. All methanol-free cultivations were performed at a constant specific growth rate ( µ = 0.065 h⁻¹) under both normoxic and hypoxic regimes, the latter implemented via automated respiratory quotient ( RQ ) control at a setpoint of 1.40. Comparative analysis indicated that only promoters associated with glycolytic metabolism (P PGK1 and P GAP ) benefited from oxygen limitation, showing increases in CalB-specific production rate ( q P ) of approximately 50% and 100%, respectively. In contrast, the novel promoters P DF and P UPP achieved the highest overall CalB expression levels, exceeding P GAP levels by more than 3-fold under hypoxic conditions and even surpassing the final titers obtained with the classical methanol-induced P AOX1 -based bioprocess. These findings highlight their strong potential for efficient, methanol-free RPP in K. phaffii. This systematic evaluation of promoter performance under physiologically controlled stress conditions provides valuable insights for maximizing RPP in K. phaffii . Moreover, the integration of precise, accurate RQ -based control underscores the potential to link physiological regulation with process design to advance next-generation, scalable biomanufacturing strategies.